This research aims to understand the molecular processes by which an allergen elicits an immune system response. In particular, we intend to advance understandings of the intracellular processes that result from antibody-induced cross-linking of the membrane receptors and induce an allergic and inflammation response. This project will advance the experimental capabilities for super-resolution microscopy through the development of an array of apertures, each illuminating a small sample volume utilizing near-field optics. The long-term goal of this research is to understand the physical and chemical processes used to modify local composition of the plasma membrane. The objective of this project is to measure the membrane reorganization and signaling processes with nano-patterened optical surfaces capable of 80 nm and 1 ps resolution with otherwise conventional fluorescent microscopy techniques. This project will yield a new technology to overcome current experimental barriers and have applications to a variety of biological hypotheses. The short-term goals of this study are (1) to develop a technique capable of single-molecule detection in concentrated biological environments and (2) to further understand IgE-FccRI clustering, protein recruitment, and the induction of local membrane heterogeneity. The central hypothesis of this research is that dielectric-filled zero-mode waveguides will provide sufficient time and space resolution on cell membranes to measure protein-protein associations induced by receptor stimulation. This hypothesis is built upon both related nanoscale structures and theoretical predictions, as described below. Such devices will provide a means for measuring membrane (re)organization, testing previously unanswerable hypotheses, and advancing the treatment of membrane-receptor based aliments. This project provides a unique training opportunity to build upon my background in biophysical chemistry, guided by mentoring in nanofabrication and membrane biology. This research will create a new technology and apply it to answer pressing questions in the intracellular signaling cascade following antigen stimulation and cross-linking of FC?RI membrane receptors. Results from this research will be directly applicable to diagnosing and treating allergic responses. Specific Aim 1: Develop an array of apertures for near-field optical microscopy (ANOMs) Specific Aim 2: Characterize the optical properties of ANOMs for quantitative super-resolution microscopy Specific Aim 3: Measure local membrane heterogeneity induced by FC?RI clustering